Inducible deletion of Raptor and mTOR from adult skeletal muscle impairs muscle contractility and relaxation

Skeletal muscle weakness has been associated with different pathological conditions, including sarcopenia and muscular dystrophy, and is accompanied by altered mTOR signaling. Here we wanted to better elucidate the functional role of mTOR on muscle contractility. Most loss of function studies for mTOR signaling have used the drug rapamycin to inhibit some of the signaling downstream of mTOR. However, as rapamycin does not completely inhibit all mTOR signaling, we generated a double k.o. for mTOR and for the scaffold protein of mTORC1, Raptor, in skeletal muscle. We found that dk.o. mice results in a more severe phenotype compared to Raptor or mTOR deletion alone. Indeed, they display muscle weakness, increased fiber denervation, and a slower muscle relaxation following tetanic stimulation. This is accompanied by a shift towards slow-twitch fibers and changes in the expression levels of calcium-related genes, like Serca1 and Casq1. Indeed, dk.o. mice show a decrease in calcium decay kinetics after tetanus in vivo, suggestive of a reduced calcium reuptake. In addition, RNA sequencing analysis revealed that many downregulated genes are linked to sarcomere organization, like Tcap and Fhod3. These results suggest a key role for mTOR signaling in maintaining a proper fiber relaxation in skeletal muscle. Overall design: Inducible, muscle-specific Raptor knockout mice were generated crossing mice expressing Raptor gene between two LoxP sites with mice carrying Cre recombinase fused to a mutated estrogen receptor domain under the control of human skeletal actin promoter. Inducible, muscle-specific Raptor mTOR double knockout mice were generated crossing HSA-Cre-Raptorfl/fl mice with mTORfl/fl mice. Tamoxifen-induced Cre LoxP recombination was activated by oral administration of tamoxifen-containing chow (Tam400/Cre-estrogen receptor, Harlan), for 7 weeks. For long-term treatment muscles were collected 7 months from the beginning of the treatment. RNA-seq was carried out on gastrocnemius muscles of wild-type, Raptor k.o. and Raptor mTOR dk.o. mice, with the contrast of interest being wt v dk.o. and k.o. v dk.o..